The overall objective of this research is to elucidate molecular integrations between leukocytes which mediate progressive accumulation and ordered packing of leukocytes at sites of evolving tissue injury and inflammation. Specific aims are (i) to identify receptors and ligands expressed by leukocytes that facilitate leukocyte-leukocyte adhesion under conditions of physiological flow, (ii) to determine specific receptor- ligand interactions between flowing or rolling leukocytes and adherent leukocytes in vitro. and (iii) to determine if inhibition of these receptor-ligand interactions abolish leukocyte accumulation in vivo. Leukocyte accumulation will be molded in vitro using a flow chamber in which purified adhesion molecules or adherent leukocytes serve as substrates for adhesion of leukocytes and transfected cell lines infused at controlled flow rates. Antibody blockade and use of transfectants differentially expressing individual adhesion molecules, such as L-selectin and P-selectin glycoprotein ligand-1, will determine the relative contribution of these molecules to leukocyte accumulation. In addition leukocytes and cell lines will be treated with enzymes to determine the role of glycosylation. sialylation and sulfation of receptor ligands mediating leukocyte-leukocyte adhesion. Finally,leukocyte accumulation in vivo will be assessed using intravital microscopy of venules in the mouse cremaster muscle. The role of specific adhesion receptors mediating leukocyte accumulation in vivo will be verified using gene-targeted mice, including mutants deficient in l,-selectin. Understanding the molecular determinants of leukocyte accumulation will provide insight into mechanisms of inflammatory tissue injury, reperfusion injury, and wound healing.